Microvillus inclusion disease (MVID) is the most severe cause of Congenital Diarrheal Diseases in neonates. MVID results from mutations that lead to malabsorption and life-threatening, intractable, secretory diarrhea. To date, no curative therapy exists. MVID patients are largely dependent on parenteral nutrition. Prognosis is generally poor, due to metabolic decompensation, dehydration, infections and liver complications associated with parenteral nutrition. The only alternative therapy to parenteral nutrition is intestinal transplantation; MVID accounts for 7% of pediatric bowel transplantation worldwide. However, the overall five year survival after small bowel transplantation is only about 50%. Parenteral nutrition and bowel transplants are non-permanent solutions for treating MVID, further work in deciphering how inactivating mutations in MYO5B lead to aberrant trafficking in enterocytes will provide novel insights into genotype-phenotype relations and pave the way for development of improved diagnosis and viable alternative therapeutic strategies. There is a limited availability of patient material and no suitable animal models for MVID, hampering the thorough understanding of the disease's molecular mechanisms. While we have recently been able to develop a mouse model of germline and intestinally-targeted deletion of MYO5B, these mice die in their first week of life, so any analysis of interventions that might alter the course f disease are not possible due to the small size. To that end, we propose utilizing our state-of-the-art gene-editing platform to develop swine with a specific mutation (P663L) corresponding to the human P660L mutation of the MYO5B gene, present in Navajo MVID patients. We hypothesize that introducing a Proline (P) to Leucine (L) mutation in pigs at a site analogous to the human P660L allele can induce MVID in piglets. Execution of the hypothesis can be accomplished with the following specific aim: develop and evaluate the MYO5B (P663L) mutant pigs as models of human MVID. In addition to state-of-the art gene editing platform with expertise of Drs. Melkamu (Veterinary Physiology) and Carlson (Animal biotechnology) from Recombinetics, we have engaged a world-renown expert in MVID (Dr. James R. Goldenring, MD, PhD; a gastroenterologist and a Professor of Experimental Surgery) from Vanderbilt University School of Medicine. A reliable large animal model of MVID will have tremendous impact on industry and academic research to develop and test new drugs and novel therapeutic approaches to treat this awful disease.